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Metagenomic Analysis of Viral Pathogens in Sewage
Overview ]] Sewage sludge is classified as a compound called a biosolid that is the direct result of accumulation of sewage wastewater. The key difference between sewage sludge and biosolids that are approved for use in agricultural applications as a fertilizer is that an approved biosolid has first been treated in a way that can make it less likely to contaminate crops with the myriad of pathogens that can be potentially found in waste products. This process is governed by the EPA, as it is environmentally preferable to reuse waste, and is tightly regulated.Water: Sewage Sludge (Biosolids) Frequently Asked Questions. United Stated EPA website. Sept. 14 2012 [http://water.epa.gov/polwaste/wastewater/treatment/biosolids/genqa.cfm Link] Sewage sludge had already been widely known to contain many various bacterial pathogens since the early 1980sDudley, D.J. et al. (1980) Enumeration of potentially pathogenic bacteria from sewage sludges. Appl Environ Microbiol. 39(1): 118–126. [http://www.ncbi.nlm.nih.gov/pmc/articles/PMC291294/ PubMed], however the complete viral profile (as complete as possible) had not previously been ascertained. To analyze the viral components, a metagenomic study was performed by Bibby and Peccia (2013) in an effort to understand the viral genomes present to potentially understand risks and potential development of better treatments for wastewater in general. To achieve this, the researchers decided to focus on the diversity of the pathogens present, as well as potentially identify other unknown pathogenic viruses that had arisen from evolution or introduction into the environment.Bibby, K., Peccia, J. (2013)Identification of viral pathogen diversity in sewage sludge by metagenome analysis. Environ Sci Technol. 47(4):1945-51. [http://www.ncbi.nlm.nih.gov/pubmed/23346855 PubMed] This is an extension of a similar project conducted by the same group in which a method for identifying these samples and a specific sequencing technique was developed with an ''in silico'' study, of which they modeled this experiment after.Bibby, K., Viau, E., Peccia, J. (2011) Viral Metagenome Analysis to Guide Human Pathogen Monitoring in Environmental Samples. Lett Appl Microbiol. 52(4):386-92. [http://www.ncbi.nlm.nih.gov/pubmed/21272046 PubMed] Analysis '''Purification and Sequencing''' The researchers in the study took wastewater samples from treatment plants located in 5 different regions of the United States (Southwest, Southeast, Midwest, and 2 Northeastern), each of which serving a population ranging from 100,000-1,000,000 individuals. From these samples, viral species as well as nucleic acids (both DNA and RNA) were purified utilizing commercially available kits. Samples were amplified using random PCR and RT-PCR amplification techniques and subjected to shotgun sequencing analysis of fragmented 200 bp segments. Sequencing data was validated by PCR analysis using primers for known human viruses. For verification purposes, PCR amplification was also conducted with known templates for viral genomes to determine if their sequencing was correct. '''Bioinformatics''' All samples were sequenced twice and resultant sequences were run through a BLAST search against the NCBI database for viral genomes. Occurrence of certain sequences within each sample was generaged through the use of a contig mapping system and calculated by dividing the log of reads by the total reads. Results and Findings A total contig count of 412,654 was obtained with 81,265 annotated in the sample. Of these annotations, 470 were identified to be pathogenic in humans. DNA/RNA for pathogens in humans that were found included a wide variety of viral families (shown in tables below) with the most prominent being that of ''Herpesvirus. ''All of the viruses shown are known to be pathogenic to humans, often with serious consequences. Relative abundancies of these viral species was calculated to gain an idea of how widespread these viruses are in the waste samples. It was found that there were four viruses (''Herpesvirus, Adenovirus, Papillomavirua, and Bocavirus'') found in 90% of samples as DNA and three as RNA (''Coronavirus, Rotovirus, and Klassevirus'') in 80% of samples. Viral sequences obtained were also found to be reproducible via PCR techniques analyzing generation of the same contiguous sequences with a high degree of accuracy. In addition, targeted PCR analysis was conducted to verify whether or not specific sequences were able to be reproduced from the expectations of the researchers, most notably ''Adenoviral ''genomic DNA, which was found in all samples. As with most genomic studies, this study was not without limitation as the number of pathogenic human viruses is quite large, all of which would be impossible to detect in a single study. In addition, verification by PCR was used to strengthen conclusions, however many viral strains and species have similar sequence idendities. This may have potentially given false readings or misidentification of certain viruses. The study was able to show a variety of pathogenic viruses that can cause both infection and respiratory disease in abundances that show how important their eradication from sludge during treatment is. The diseases that are possibly related to infection of these viruses from waste can range from latent infections such as herpes, to viruses that can lead to aggrivated infection of the airways. These findings show that there needs to be more care taken in not only processing to prevent infection, but spreading of sludge in agricultural proceses given the high abundance of respiratory pathogens.Additionally, 4 viruses previoulsly not found in sludge analyses (Parechovirus, lassevirus, Bocavirus, and Coronavirus) were identified as emerging in the viral profile of such environments, leading to the conclusion that there may need to be revisions made to the current treatment procedures and regulations. References